Customary immunological methods for diagnosing diseases which are accompanied by the formation of specific antibodies against a disease elicitor, such as viruses, bacteria, allergens, autoantigens or particular pharmaceuticals, are based on the ability of these antibodies to form complexes with antigenic structures belonging to the elicitor.
In particular embodiments of these methods, which are generally termed heterogeneous immunoassays, a sample which is to be examined for its content of, for example, specific antibodies (analyte antibodies) is brought into contact with antigenic structures of the disease elicitors, with these antigenic structures being immobilized on suitable, known support materials. Analyte antibodies which are present in the sample are bound, as an immune complex, to the antigenic structures of the disease elicitor which are immobilized on the support materials, and detected. Detection antibodies or other specific receptors (e.g. protein A) which are capable of complexing with the analyte antibody of the sample can be used for the detection.
As a rule, the detection reagent carries a label which makes it methodologically possible to detect the quantity of the antibody which is bound.
Commonly used labels are radioactive isotopes, enzymes, fluorescent, phosphorescent or luminescent substances, substances having stable unpaired electrons, latex particles, magnetic particles, metal sols and erythrocytes.
These methods are known to include both single-step and multistep detection methods. Each procedural step is customarily terminated by a separation process (washing step).
However, in heterogeneous immunoassays, the technique of the single-step method, which technique is very simple to carry out, is not suitable for detecting all disease markers. Two-step or multistep methods frequently have to be employed for technical reasons.
However, multistep methods, which are termed immuno-complex transfer enzyme immunoassays (S. Hashida et al., Journal of Clinical Laboratory Analysis 8:86-95 (1994)), are also known. In these methods, the entire immune complex, comprising solid phase antigen, specific antibody and labeled conjugate antigen, is detached from the solid phase. The entire immune complex is then fixed and detected after having been transferred by pipette to an antibody-binding solid phase.
While these methods are very specific, they suffer from the disadvantage that the disease elicitors to be detected or antibodies directed against them which have entered, in the first step, into a complex with the immobilized, specific receptor, can, in a reverse reaction which is known to the skilled person, in part become detached again from the complex in the subsequent reaction steps and consequently evade the detection reaction, resulting in the sensitivity, for example, being reduced.
The diagnostic efficiency of such multistep methods is reduced to a particularly great extent when the rate of the reverse reaction between the immobilized receptor and the agent to be detected is high. Such a high rate is obtained, for example, in the case of low-affinity antibodies against disease elicitors or pharmaceuticals. The skilled person knows that these effects are obtained, in particular, in the case of methods for detecting frequently mutating disease elicitors or disease markers which, following mutation, exhibit only slight interaction with the immobilized specific receptor.
EP 0 572 845 has disclosed that the reverse reaction rate is substantially reduced by adding a further receptor against structural features of the agent to be detected. This receptor has to possess more than one binding site for the agent to be detected and must not interfere with the immunochemical detection of the agent.
However, despite a marked reduction in the reverse reaction, it is likewise not possible to use this method to detect special low-affinity antibodies against disease elicitors or pharmaceuticals reliably and at high sensitivity.
The object was, therefore, to find reagents which do not have these disadvantages.